Tuberculosis (TB) is an important health concern for veterans and service members who serve in TB-endemic countries. There is an urgent need for new shorter TB treatment. The length of treatment required to prevent relapse is determined by the effectiveness of drugs during the long ?sterilizing? phase of treatment that follows early bactericidal killing. Different tissue micro-environments harbor M. tuberculosis (Mtb) populations with different replication rates that respond differently to drug treatment. For example, drugs are more effective against M. tuberculosis (Mtb) in airway and aerated cellular lung infiltrates than in the caseous hypoxic core of necrotic granulomas. We have pioneered a suite of novel assays that will enable us to probe fundamental characteristics of Mtb physiologic state in tissues for the first time. These assays provide an unprecedented lens with which to re-examine historical assumptions about the influence of tissue micro-environment on drug response. These novel assays also enable us to test a novel alternative paradigm for drugs' treatment shortening (sterilizing) activity. Specifically, our novel construct is that reduction in Mtb burden during the sterilizing phase is the combined result of two distinct processes: (1) killing and (2) inhibition of replication. The first process ? killing ? has long been the primary focus of TB drug development. We hypothesize that the second process ? abrogation of Mtb replication ? is an important but previously unrecognized contributor to drug sterilizing activity. We believe that drug inhibition of replication in the sterilizing phase of treatment shortens the time needed for durable, non-relapsing cure. We will test this in the C3HeB/FeJ (Kramnik) mouse, a strain that develops well- encapsulated necrotic granulomas with low internal oxygen tension. This model enables us to study Mtb in a spectrum of micro-environments that mimic the pathology of human TB. We will measure Mtb rRNA synthesis at three points (baseline, bactericidal and sterilizing phases) in: (1) airway, (2) cellular lesions, (3) lymphocytic outer cuff of granulomas and (4) hypoxic caseum of necrotic granulomas. Using several complementary methods to quantify Mtb burden, we will determine specifically where Mtb is cleared and where Mtb persists. To test our hypothesis that inhibition of replication contributes to treatment shortening, we will compare regimens with different sterilizing potency: standard treatment (isoniazid, rifampin, pyrazinamide, ethambutol [HRZE]) and a novel regimen that cures TB substantially faster (bedaquiline, pretomanid, linezolid, pyrazinamide [BPaLZ]). Aim 1 will test the association between tissue micro-environmental conditions and Mtb replication before treatment. We hypothesize a spectrum of Mtb rRNA synthesis and replication, ranging from fastest in airway, intermediate in cellular lesions and lymphocytic cuff to slowest in the caseum. Aim 2 will test the association between Mtb replication and drug killing during the bactericidal phase. We hypothesize that drugs will preferentially kill Mtb in micro-environments where Mtb is replicating rapidly (i.e., airway, lymphocytic cuff and cellular lesions). There will be minimal killing of Mtb in caseum during the bactericidal phase. Aim 3 will evaluate the sterilizing phase. Aim 3 will test our hypothesis that a highly potent sterilizing regimen (BPaLZ) will both clear Mtb more effectively and suppress Mtb rRNA synthesis and replication more profoundly than the standard regimen (HRZE). This work will contribute to development of a practical biomarker of sterilizing activity. A marker of sterilizing activity will lead to shorter treatment by improving preclinical drug assessment and improving the accuracy and efficiency of drug trials. Ultimately, this program will also lead to a clinical biomarker for routine monitoring to guide duration of treatment among veterans treated for TB. Our approach could be further extended to many other chronic bacterial infections that are important for veterans.